Fig. 3: Differential requirement of Tse5-CT and Tse5-NT cleavage for toxin activity.

a Structural superposition of Tse5 (grey), RhsA (orange), and Rhs1 (yellow) N- and C-terminal plugs. N- and C- terminal residues (P48 and L1168) in Tse5’s N- and C-terminal plugs are indicated. Conserved aspartic residues in the aspartyl protease motif within the C-terminal plug are also indicated (D1141 and D1164). b Conservation in the N- and C-terminus of Tse5-Shell. Sequence logo around Tse5 residues 45–84 (top) and 1135–1172 (below) generated from an alignment of 2225 sequences from Pseudomonas containing the N-terminal plug domain of unknown function (DUF6531; InterPro entry PF20148). Tse5 cleavage sites are indicated with a dotted line. Mutated positions that inhibit the N-terminal and C-terminal cleavage in Rhs1 (black *), RhsA (red *), TseI (green *) or Tse5 (red *) are starred. Sequence conservation consensus and the Tse5 sequence are provided below. Sequence logo generated by WebLogo⁷⁸. c N- and C-terminal cleavage sites. The Tse5-Shell is depicted in grey/white cartoon. In the upper panel, cleavage site of the C-terminus (between L1168 and I1169) and the catalytic aspartic residues (D1141 and D1164) are shown. The lower panel illustrates the N-terminal cleavage site (between K47 and P48). d Representative SDS-PAGE of purified Tse5 and variants. Similar results were obtained for >10 protein batches purified to homogeneity. The Tse5-NT (green arrow) and Tse5-CT (red arrow) fragments are visible in the pure Tse5. Mutation of the N-terminal cleavage site (Tse5-K47G-P48A) abrogates the scission of the 10-kDa Tse5-NT. Mutation of the catalytic aspartic residues (D1141A and D1164A) abrogates the scission of the ~16-kDa Tse5-CT.